Recombinant DNA technology has made it possible to clone and amplify DNA fragments from the chromosomes of high eucaryotes (plants and animals) using vectors that can be propagated in bacteria such as Escherichia coli (E. coli).
If DNA is to be introduced directly into bacteria that have been made competent for that uptake, then the DNA must be relatively small (less than 20 kb) since the efficiency of DNA uptake into cells decreases dramatically as the size of the DNA increase above 20 kb. Accordingly, alternate delivery routes have been sought in order to introduce large DNA into bacteria. The technique of packaging bacteriophage vector DNA into virus particles was developed to meet this need. It has the advantage of delivering the packaged DNA to cells by infection with near unit efficiency. Bruning et al., Gene 4, 85-107 (1978) and Collins et al., Proc. Natl. Acad. Sci., 75, 4242-4246 (1978) took advantage of the in vitro packaging reaction of bacteriophage lambda, developed by Sternberg et al., Gene 1, 255-280 (1975) to package large inserts cloned into cosmid vectors which are fusions of a plasmid and a bacteriophage lambda cos site. The cos site provides the recognition element needed to initiate the packaging of DNA into a lambda bacteriophage head.
The major disadvantage of the lambda bacteriophage in vitro packaging reaction is that the lambda bacteriophage head cannot accommodate more than 49.5 kb of DNA. Thus, taking into account that the cos vector itself is about 2 kb in size, then no more than 47 kb of clonable DNA can be inserted into the vector and packaged into a lambda bacteriophage head. (Murray, Lambda II 236, 395-432, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1986).
Accordingly, the lambda phage cannot be used to clone genes bigger than 50 kb, and mapping chromosomal segments of DNA that are several megabases in size by "walking" or "jumping" protocols is difficult, given the limitations of the lambda bacteriophage system. Researchers have explored two systems as potential tools for cloning larger DNA fragments: phage systems with head sizes larger than bacteriophage lambda and yeast cloning vectors.
Rao et al., J. Mol. Biol. 185, 565-578 (1985), demonstrated that T4 DNA (about 165 kilobases) could be packaged into bacteriophage T4 heads in vitro. The efficiency of that reaction is 10.sup.4 -10.sup.5 plaque-forming units (PFU) per microgram of added T4 DNA. However, Black, Gene 46, 97-101 (1986), showed that efforts to package vector DNA of various sorts into T4 heads and recover that DNA following injection into appropriate bacteria was no more efficient than 10.sup.2 -10.sup.3 PFU per microgram of added DNA. Black's result suggests that it may be very difficult to use the T4 packaging system to generate a complete library of 150 kb inserts of a complex genome such as that of mammalian cells (i.e., the equivalent of about 20,000 inserts of 150 kb). A second, and perhaps more difficult problem, is the absence of a cloning vector that can take advantage of the T4 packaging capability to clone and amplify large DNA fragments. Indeed, since very little is known about the T4 sequences that are needed to initiate T4 packaging, it may be difficult to construct such a vector.
Burke et al., Science 236, 806-812 (1987), were able to clone large segments of mammalian DNA as minichromosomes in yeast. The DNA to be inserted is cloned into a vector containing a yeast replicating element, a yeast partitioning element or centromere, and yeast telomeres and the resulting chimeric DNA is introduced into yeast by direct DNA transformation. Minichromosomes with inserts of DNA larger than 100 kb were identified. There are two problems with this system. First, yeast clones with inserts of DNA are generated very inefficiently; in the experiments described, about 300 clones were produced per microgram of insert DNA. Second, once clones with inserts of DNA are available, it is difficult to probe and recover the insert DNA. In transformed yeast cells, minichromosomes represent less than 1:200 of the total DNA of the cell and consequently can only be detected by DNA hybridization techniques. Moreover, within each transformed cell the inserts cannot be amplified. Segments of the insert can be recovered by subcloning into plasmids and rescuing into bacteria.
Another system of interest is described by Gaitanaies et al., Gene 46, 1-11 (1986). A lambda vector is described into which DNA can be inserted and which can then be injected into cells. The injected DNA is maintained in cells either integrated into the host's chromosome at one copy per cell or extrachromosomally in multiple copies per cell. While this vector can accommodate less than 30 kb of DNA, the amount of DNA in the integrated prophage can be increased significantly by homologous recombination with a second infecting lambda-chimera whose insert overlaps that in the integrated prophage. In this way the segment of contiguous DNA in any prophage can be increased to more than 100 kb and subsequently amplified by inducing the extrachromosomal state of the vector. However, to accomplish this, one needs to have cloned at least several contiguous and overlapping smaller segments of DNA and carry out an arduous process of stringing them together by recombination. Moreover, it will be difficult to recover the large DNA insert once it is constructed, since it will be too big to be packaged into a lambda virus-particle. Direct isolation of the DNA in its extrachromosomal state could also be difficult because of its large size.
Recently, Blumenthal, Focus, pages 41-46, Vol. 11, No. 3 (Summer 1989), reviewed some of the difficulties relating to cloning and restriction of methylated DNA in E. coli. The remedy suggested on page 45 is that such cloning (of methylated DNA) should be carried out in a host lacking the three known methylation dependent restriction systems including both components of the McrB system.